Secondary label-conjugated and non-conjugated detection antibodies are frequently used in a wide range of research applications. However, they are often affinity-isolated, polyclonal reagents that may lack the highest standard of antibody validation. The antibodies characterised in this study are a polyclonal anti-chicken IgY antibody produced in rabbit (31104, Thermo Fisher) and a polyclonal goat anti-rabbit IgG antibody conjugated with alkaline phosphatase (AP) (A3687, Sigma-Aldrich). Although the use of the rabbit anti-IgY antibody in the literature is limited, the goat anti-rabbit IgG AP was extensively utilised in research for over 15 years ^{1, 2} .

The research conducted in this laboratory examines complex antibody repertoires in humans and animals by means of protein arrays. Protein arrays are frequently used to profile antibody binding to human proteins in autoimmune disease ³ , cancer ⁴ and in healthy individuals ⁵ . Other protein array applications include recombinant ⁶ and hybridoma-derived ⁷ antibody characterisation studies. This article investigates the cross-reactivity of a rabbit anti-chicken IgY and an alkaline phosphatase-conjugated goat anti-rabbit IgG, which were used for the profiling of IgY antibody responses to human antigens in chickens immunised with human cancer cells. The protein array technology applied here, developed by Büssow and colleagues ⁸ , is comprised of a fully annotated set of 7,390 distinct human proteins, in its current version, that may serve as potential antigens. The aim of this study is to define a cross-reactivity reference list for the two described secondary antibodies, which can then be used to eliminate non-specific binders from ongoing chicken IgY profiling studies. Furthermore, publication of the cross-reactivity reference list may support other researchers using these antibodies in the evaluation of their experiments.

Probing protein arrays with antibodies enables the assessment of specificity and cross-reactivity on large numbers of potential antigens in parallel. Here we investigated the cross-reactivity of secondary anti-chicken IgY from rabbit and anti-rabbit IgG AP from goat using human protein arrays in the absence of chicken serum. The analysis revealed antibody binding to human proteins in the absence of chicken serum and hence chicken IgY immunoglobulins. The identified positive signals varied in strength, as shown in Figure 1, with intensity 3 being the strongest and 1 the weakest. The difference in signal intensities may relate to varying protein quantities on the array and differences in antibody affinities to corresponding proteins. A total of 63 binding events were visible on the protein arrays, of which 61 corresponded to unique proteins ( Table 4). Five of the identified signals were scored as intensity 3, twelve signals were scored as intensity 2 and remainder were scored as intensity 1. The original protein array images are shown in Figure S1 and Figure S2 ( Supplementary material) and protein array images with highlighted positive signals, which correspond the cross-reactive proteins listed in Table 4, are shown in Figure S3 and Figure S4 ( Supplementary material).

The investigated antibodies were found to bind to a wide range of human proteins ( Table 4). However, it is worth noting that a total of six identified binding events correlated to human immunoglobulin proteins, with four scored at the highest intensity (Intensity 3). Such cross-reactivity is not surprising considering the antibodies are polyclonal and the immunogens of both hosts were immunoglobulins. In addition, the data sheet provided with the anti-chicken IgY antibody produced in rabbit (31104, Thermo Fisher) has specified that this antibody may cross-react with immunoglobulins from other species. The data sheet for the goat anti-rabbit IgG AP antibody (A3687, Sigma-Aldrich) has specified binding to all rabbit immunoglobulins.

This work illustrates the cross-reactivity of an antibody-based detection system for IgY binding. The polyclonal anti-IgY rabbit antibody in combination with an anti-rabbit IgG alkaline phosphatase-conjugated antibody was shown to bind to 61 human proteins present on Unipex protein arrays comprising of 7,390 human proteins. Characterisation of this cross-reactivity provides a ‘false-positive’ database for future chicken antisera characterisation on protein array systems not limited to the Unipex protein array used here. These results, in combination with ‘false-positives’ from earlier research investigating antibody cross-reactivity by this group ⁹ and others ¹⁰ may provide valuable information for future protein array-based experiments. Reference lists provided by such experiments would be further strengthened by arrays that include additional portions of the human proteome and/or post-translational modifications. Using antibodies that have been extensively characterised on protein arrays will reduce the risk of identifying irrelevant cross-reactive secondary antibody binding to the array as a host-antigen response.

Overall, the antibodies tested here showed cross-reactivity to unrelated human proteins as well as to human immunoglobulin proteins, which are homologous to the original immunogens. Despite the identified non-specific binding, the tested antibodies are suitable for use in protein array experiments as the cross-reactive binding partners can be readily excluded from further analysis. As both antibodies were used as a pair in this study, the possibility to deduce the exact cross-reactivity profile for each individual antibody may be limited. However, the cross-reactivity reference list provided in this paper can be further utilised to validate research using those antibodies in applications other than protein arrays.